GSM7178444: Hi-C_NeuN+_H4; Homo sapiens; Hi-C
Post-mortem human brain samples were obtained via National BioService Russian Biospecimen CRO (St. Petersburg, Russia). Informed consent for using human tissues for research was obtained in writing form from all donors or their next of kin by the tissue provider bank. Sampled brain tissue of all subjects was defined as healthy by medical pathologists. All subjects had sudden death with no prolonged agony state and had no history of neurologic and psychiatric disorders or alcohol and drug abuse. Post-mortem brain frontal blocks 1-1,5 cm thick were stored at -80°C warped in a foil, in zip-lock packages. For sample dissection, The Atlas of the Human Brain (AHB) (Mai 2016) was used to locate the area of interest, a posterior part of the superior temporal gyrus of the left hemisphere (posterior Brodmann area 22 (BA22p), Wernicke's area). Frontal blocks with surfaces nearest to AHB level: 60 (MNI: -36.57; ICL: 34.82; MCP: 20.08) were placed into the cryostat Leica CM1950 chamber with -20°C before dissection for 15-20 min on custom-made aluminum support. A tissue piece of approximately 150 mg weight was cut out from a selected area using a metal scalpel. Dissected samples were then collected with tweezers, put into tubes, and placed in dry ice immediately. All materials used during dissection (scalpels, tweezers, tubes) were sterile and chilled using dry ice before use. Preparation of bulk Hi-C libraries from 11 samples of human brain tissue was performed as previously described (Lieberman-Aiden et al. 2009; Ulianov et al. 2016) with optimal changes to brain tissue (Eremenko et al. 2021). Approximately 200,000-400,000 sorted nuclei for the sample were lysed in a solution of lysis buffer containing 150 mM Tris-HCI pH7.5-8, 150 mM NaCI, 0.5% (v/v) NP-40, 1% (v/v), Triton-X100, 1x Protease Inhibitor (CALBIOCHEM, #539137) on ice for 15 min, centrifuged at +4 °C 5000g for 7 min, the pellet was resuspended and washed in 1.12Х restriction enzyme DPN II buffer. After the final wash, the pellet suspension in 1.12Х DPN II buffer with added 0.3% SDS has incubated for 60 min 1400 rpm at +37°C. Further, the reaction was quenched with 1.8% Triton X-100 for 60 min at 1400 rpm at +37°C, and the 500U DPN II restriction enzyme was added, the suspension was left overnight at 1400 rpm at +37°C. After that, the additional amount of 200U DPN II restriction enzyme was added to the digested nuclei and incubated for the next 120 min at 1400 rpm at +37°C. After that, the restriction was unactivated at +65°C for 20 min. The digested DNA samples were washed and resuspended in 1.2XNEB2, and after that digested ends were marked with biotin during the incubation in the mix of 1М biotin-14dATP, 10x NEB2, 10mM dCTP, 10mM dGTP, 10mM dTTP, 5U/ul Klenow (NEB #M0210S) for 90 min 900 rpm at +37 °C. The resulting blunt ends were washed with 1X T4 DNA ligase buffer and chromatin fragments were proximally ligated with 50 U T4 DNA ligase (Fermentas) for 6 h 1400 rpm at +37 °C. Then the 0.5% SDS with 20 μg/ml proteinase K was added to samples for overnight incubation at +65 °C. The following step was DNA purification with phenol–chloroform extraction and precipitation with 2.5 volumes of 96% ethanol, 0.1 volumes of 3M sodium acetate, 20 μg/ml glycogen, and 9.4 μg/ml tRNA for 60 min at −80 °C. After the centrifugation step at 20000g for 20 min, +4°C the pellet was dissolved in 10 mM Tris-HCl pH 8.0 with the addition of RNase A and incubated for 45 min at +37°C. The next step was DNA sample purification on 2.5 volumes of magnetic AMPure XP beads with the following DNA elution in 10 mM Tris-HCl for 15 min at +55°C. Eluted DNA was fragmented by sonication with Power 15 regime, 4 repeats for 30 s with 3 min break. Sonicated DNA samples were then concentrated on Amicon columns 14000g/5 min with double 10 10 mM Tris-HCl pH 8.0 washing. The resulting biotin-marked fragments were isolated using Dynabeads MyOne Streptavidin C1 (Invitrogen #65001) and diluted in TWB buffer (1M Tris-HCl pH 8.0, 50 mM EDTA, 5M NaCl, 1% Tween). All subsequent steps were performed on that bead-bound DNA fraction including DNA ends repair with repair mix (10Х ligase NEB buffer, 10 mM dNTP mix, T4 DNA Pol, T4 PNK, 1 ul Klenow) for 30 min at room temperature, and PolyA tail adding reaction with a mix containing 10Х NEB2, 10 mМ dATP, Klenow (exo-) and incubation for 30 min at +37°C and then for 20 min at +65°C. Then beads were washed with 1X T4 ligase buffer for 3 min at room temperature 900 rpm, and the ligation reaction was performed with a ligation mix of 10X T4 ligase buffer, adapter, 5 U T4 ligase (Fermentas) for 2.5 h at room temperature. Finally, libraries were PCR amplified using Illumina  forward  and reverse primers and KAPA HiFi HotStart for 16 cycles to select the appropriate cycle for amplified DNA fragments collection (the 6, 9, 12, and 15 cycles were collected and checked for DNA quantity on chosen cycle). Last, up to the chosen number of cycles Hi-C libraries were PCR amplified, and resulting fragments were purified using 1.5 volumes AMPure XP magnetic beads.